Green algal hydrogenase activity is outcompeted by carbon fixation before inactivation by oxygen takes place.

Photoproduction of hydrogen by green algae is considered a transitory release valve of excess reducing power and a potential carbon-free source of sustainable energy. It is generally accepted that the transitory production of hydrogen is governed by fast inactivation of hydrogenase by oxygen. However, our data suggest that photosynthetic electron loss to competing processes, mainly carbon fixation, stops hydrogen production, supports hydrogen uptake, and precedes the inevitable inactivation by oxygen. Here, we show that when transitioning from dark anaerobiosis to light, hydrogen production ceases within 2 min, regardless of the presence of oxygen. Simultaneous monitoring of the active hydrogenase pool size shows that it remains entirely intact up to 4 min after illumination and is inactivated only later. Thus, our data reveal a window of 4 min in which the hydrogenase pool is not being degraded by oxygen. Furthermore, we show that electron loss, prominently to carbon fixation, outcompetes hydrogen production and leads to hydrogen uptake. Indeed, supplying additional reducing power to hydrogenase at the cessation point regenerates the accumulation of hydrogen. Our results imply the fast cessation of hydrogen production is governed by electron loss rather than oxygen inactivation, which takes place minutes later.